15
Virology (MD PhD) (Subject) / Huber (Lesson)
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- DNA microarrays - High density DNA array - Millions of different oligonucleotides spotted onto a solid support - Labeled sample DNA (e.g. biotinylated) is added and allowed to bind - Bound sample DNA is detected using a fluorescent probe - Used to detect viruses, and also point mutations
- Enzyme-Linked Immunosorbent Assay (ELISA) - ELISA allows the detection of viral antigen or antibody, using a solid-phase assay system Antigen ELISA (Antibody < viral Antigen < Indicator Antibody- Antigen is immobilized on plate by capture antibody - Viral antigen is detected Antibody ELISA (Antigen < Antibody in sample < Indicator Antibody- Viral antigen is immobilized on plate - Antibody directed against a specific virus protein is detected - Serology/measurement of antibody titer
- Immunoblotting (Western blot) - Detect specific viral proteins in lysates from infected tissues or cells - Detect labeled antibodies against viral proteins - Used in research and diagnostics - eg Inno-Lia HIV test (gp120/41, p24, p17...)
- Hemagglutination assay Measure of virus particles- Many viruses contain proteins that bind red blood cells (erythrocytes)- eg influenza virus hemagglutinin (HA) protein binds to sialic acid residues on the red blood cells Assay principle:- Normally, red blood cells will fall to the bottom of a culture well, forming a sharp dot - If viruses are present, they cross link red blood cells via binding to sialic acid.- A lattice of cells forms that coats wells (no dot visible). - The assay is fast (30 minutes).
- Nested PCR - Two successive runs of PCRs to increase sensitivity (reduces non-specific binding in products due to the amplification of unexpected primer binding sites) - The second set of primers amplifies a secondary target within the first round product
- Real time PCR with probes 1. Polymerization: During the annealing stage of the PCR both probe and primers anneal to the DNA target. The close proximity of the reporter to the quencher prevents detection of its fluorescence. Polymerization of a new DNA strand is initiated from the primers up to the point where the polymerase reaches the probe. 2. Strand displacement: the polymerase reaches the probe and starts displacing it. 3. Cleavage: the 5'-3'-exonuclease of the polymerase degrades the probe. This leads to a physical separation of the fluorescent reporter from the quencher. Now the probe starts emitting fluorescence, resulting in an increase in fluorescence. - Fluorescence is detected and measured in a real-time PCR machine. - An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
- Real time PCR: Quantification 1. Measure serial dilution of DNA target with known concentration 2. Prepare standard curve: (genome copies/ml versus ct-value) 3. Calculate DNA concentration of samples low ct = high concentration
- Viral enzyme assays Measure the activity of virally-encoded enzymes - reverse transcriptase (retroviruses, hepatitis B virus) - viral proteases - Product-Enhanced Reverse Transcriptase (PERT) Assay
- Measuring virus replication Animal culture (in vivo infection systems)- Used today to investigate host-pathogen interactions, vaccine and drug efficacy Embryonated eggs - Still used to generate highly concentrated virus, e.g. for Influenza vaccine production- A wide variety of viruses can grow, eg orthomyxo, paramyxo, rhabdo, toga, herpes and pox - Provides high yield of virus Cell culture methods (in vitro infection systems)- Most widely used in research today - Use in diagnostics declining
- Cell cultures 1. Primary Cell Culture- A primary cell culture is defined as a culture of cells obtained directly from the original tissue and that has been cultivated in vitro for the first time. - Some viruses only grow in primary cells. 2. Cell lines- Immortalized cell lines, sub-cultured for many passages. - Culturing in cell lines can however lead to adaptation (sequence changes, e.g. HIV-1 T-cell line adapted strains). 3. Reporter cell lines - Contain a reporter gene, which expresses marker upon infection, e.g. green fluorescent protein, luciferase oder b-gal under LTR control as indicator for HIV infection
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- TCID50 calculation by Reed-Muench Takes the proportional distance between the percent infected of the dilution before and the dilution after and the actual TCID50 in to account
- TCID50 modification by Spearman and Kaerber - More robust than Reed and Muench - Considers all events
- Multiplicity of Infection (MOI) = Ratio of the number of virus particles to the number of target cells - Multiplicity of infection can be expressed using any quantitative measure of virus titer.– particles/cell – TCID50/cell – PFU/cell – HA units/cell - MOI differs depending on cell type, culturing conditions etc.
- Virus titration: plaque assay - Takes advantage of cytophatic effect of viruses - Solid cell monolayer is infected with 10-fold dilutions of a virus stock - Virus is allowed to attach, then the cell monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. - The spread of progeny viruses during culturing is restricted to neighboring cells by the gel. - Each infectious particle produces a circular zone of infected cells called a plaque. - Eventually the plaque becomes large enough to be visible to the naked eye (crystal violet dye) - different viruses have different particle:PFU (plaque forming unit) ratios!
- Neutralization assays – Antibodies are added to a virus preparation, and the infectivity of this preparation is then measured using indicator cells. Neutralization assays served to define viral serotypes– poliovirus serotypes were defined serologically using neutralization assays (i.e., poliovirus type 1 is neutralized by antiserum to type 1 viruses, but not by antiserum to types 2 or 3 etc).